A number of procedures are presently available for the detection of specific nucleic acid molecules. These procedures typically depend on sequence-dependent hybridisation between the target nucleic acid and nucleic acid probes which may range in length from short oligonucleotides (20 bases or less) to sequences of many kilobases (kb).
The most widely used method for amplification of specific sequences from within a population of nucleic acid sequences is that of polymerase chain reaction (PCR) (Dieffenbach, C and Dveksler, G. eds. PCR Primer: A Laboratory Manual. Cold Spring Harbor Press, Plainview N.Y.). In this amplification method, oligonucleotides, generally 20 to 30 nucleotides in length on complementary DNA strands and at either end of the region to be amplified, are used to prime DNA synthesis on denatured single-stranded DNA. Successive cycles of denaturation, primer hybridisation and DNA strand synthesis using thermostable DNA polymerases allows exponential amplification of the sequences between the primers. RNA sequences can be amplified by first copying using reverse transcriptase to produce a complementary DNA (cDNA) copy. Amplified DNA fragments can be detected by a variety of means including gel electrophoresis, hybridisation with labelled probes, use of tagged primers that allow subsequent identification (eg by an enzyme linked assay), and use of fluorescently-tagged primers that give rise to a signal upon hybridisation with the target DNA (eg Beacon and TaqMan systems).
As well as PCR, a variety of other techniques have been developed for detection and amplification of specific nucleotide sequences. One example is the ligase chain reaction (Barany, F. et al., Proc. Natl. Acad. Sci. USA 88, 189-193, 1991).
Another example is isothermal amplification which was first described in 1992 (Walker G T, Little M C, Nadeau J G and Shank D. Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system. PNAS 89: 392-396 (1992) and termed Strand Displacement Amplification (SDA). Since then, a number of other isothermal amplification technologies have been described including Transcription Mediated Amplification (TMA) and Nucleic Acid Sequence Based Amplification (NASBA) that use an RNA polymerase to copy RNA sequences but not corresponding genomic DNA (Guatelli J C, Whitfield K M, Kwoh D Y, Barringer K J, Richmann D D and Gingeras T R. Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication. PNAS 87: 1874-1878 (1990): Kievits T, van Gemen B, van Strijp D, Schukkink R, Dircks M, Adriaanse H, Malek L, Sooknanan R, Lens P. NASBA isothermal enzymatic in vitro nucleic acid amplification optimized for the diagnosis of HIV-1 infection. J Viral Methods. 1991 December; 35(3):273-86).
Other DNA-based isothermal techniques include Rolling Circle Amplification (RCA) in which a DNA polymerase extends a primer directed to a circular template (Fire A and Xu S Q. Rolling replication of short circles. PNAS 92: 4641-4645 (1995), Ramification Amplification (RAM) that uses a circular probe for target detection (Zhang W, Cohenford M, Lentrichia B, Isenberg H D, Simson E, Li H, Yi J, Zhang D Y. Detection of Chlamydia trachomatis by isothermal ramification amplification method: a feasibility study. J Clin Microbiol. 2002 January; 40(1):128-32.) and more recently, Helicase-Dependent isothermal DNA amplification (HDA), that uses a helicase enzyme to unwind the DNA strands instead of heat (Vincent M, Xu Y, Kong H. Helicase-dependent isothermal DNA amplification. EMBO Rep. 2004 August; 5(8):795-800.)
Recently, isothermal methods of DNA amplification have been described (Walker G T, Little M C, Nadeau J G and Shank D. Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system. PNAS 89: 392-396 (1992). Traditional amplification techniques rely on continuing cycles of denaturation and renaturation of the target molecules at each cycle of the amplification reaction. Heat treatment of DNA results in a certain degree of shearing of DNA molecules, thus when DNA is limiting such as in the isolation of DNA from a small number of cells from a developing blastocyst, or particularly in cases when the DNA is already in a fragmented form, such as in tissue sections, paraffin blocks and ancient DNA samples, this heating-cooling cycle could further damage the DNA and result in loss of amplification signals. Isothermal methods do not rely on the continuing denaturation of the template DNA to produce single stranded molecules to serve as templates from further amplification, but on enzymatic nicking of DNA molecules by specific restriction endonucleases at a constant temperature, or unwinding the DNA duplex by the use of helicase enzymes.
The technique termed Strand Displacement Amplification (SDA) relies on the ability of certain restriction enzymes to nick the unmodified strand of hemi-modified DNA and the ability of a 5′-3′ exonuclease-deficient polymerase to extend and displace the downstream strand. Exponential amplification is then achieved by coupling sense and antisense reactions in which strand displacement from the sense reaction serves as a template for the antisense reaction (Walker G T, Little M C, Nadeau J G and Shank D. Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system. PNAS 89: 392-396 (1992). Such techniques have been used for the successful amplification of Mycobacterium tuberculosis (Walker G T, Little M C, Nadeau J G and Shank D. Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system. PNAS 89: 392-396 (1992), HIV-1, Hepatitis C and HPV-16 Nuovo G. J., 2000), Chlamydia trachomatis (Spears P A, Linn P, Woodard D L and Walker G T. Simultaneous Strand Displacement Amplification and Fluorescence Polarization Detection of Chlamydia trachomatis. Anal. Biochem. 247: 130-137 (1997).
The use of SDA to date has depended on modified phosphorthioate nucleotides in order to produce a hemi-phosphorthioate DNA duplex that on the modified strand would be resistant to enzyme cleavage, resulting in enzymic nicking instead of digestion to drive the displacement reaction. Recently, however, several “nickase” enzyme have been engineered. These enzymes do not cut DNA in the traditional manner but produce a nick on one of the DNA strands. “Nickase” enzymes include N.Alw1 (Xu Y, Lunnen K D and Kong H. Engineering a nicking endonuclease N.Alw1 by domain swapping. PNAS 98: 12990-12995 (2001), N.BstNB1 (Morgan R D, Calvet C, Demeter M, Agra R, Kong H. Characterization of the specific DNA nicking activity of restriction endonuclease N.BstNBI. Biol Chem. 2000 November; 381(11):1123-5.) and Mly1 (Besnier C E, Kong H. Converting Mly1 endonuclease into a nicking enzyme by changing its oligomerization state. EMBO Rep. 2001 September; 2(9):782-6. Epub 2001 Aug. 23). The use of such enzymes would thus simplify the SDA procedure.
In addition, SDA has been improved by the use of a combination of a heat stable restriction enzyme (Ava1) and Heat stable Exo-polymerase (Bst polymerase). This combination has been shown to increase amplification efficiency of the reaction from a 108 fold amplification to 1010 fold amplification so that it is possible, using this technique, to the amplification of unique single copy molecules. The resultant amplification factor using the heat stable polymerase/enzyme combination is in the order of 109 (Milla M. A., Spears P. A., Pearson R. E. and Walker G. T. Use of the Restriction Enzyme Ava1 and Exo-Bst Polymerase in Strand Displacement Amplification Biotechniques 1997 24:392-396).
To date, all isothermal DNA amplification techniques require the initial double stranded template DNA molecule to be denatured prior to the initiation of amplification. In addition, amplification is only initiated once from each priming event.
For direct detection, the target nucleic acid is most commonly separated on the basis of size by gel electrophoresis and transferred to a solid support prior to hybridisation with a probe complementary to the target sequence (Southern and Northern blotting). The probe may be a natural nucleic acid or analogue such as peptide nucleic acid (PNA) or locked nucleic acid (LNA) or intercalating nucleic acid (INA). The probe may be directly labelled (eg with 32P) or an indirect detection procedure may be used. Indirect procedures usually rely on incorporation into the probe of a “tag” such as biotin or digoxigenin and the probe is then detected by means such as enzyme-linked substrate conversion or chemiluminescence.
Another method for direct detection of nucleic acid that has been used widely is “sandwich” hybridisation. In this method, a capture probe is coupled to a solid support and the target nucleic acid, in solution, is hybridised with the bound probe. Unbound target nucleic acid is washed away and the bound nucleic acid is detected using a second probe that hybridises to the target sequences. Detection may use direct or indirect methods as outlined above. Examples of such methods include the “branched DNA” signal detection system, an example that uses the sandwich hybridization principle (1991, Urdea, M. S., et al., Nucleic Acids Symp. Ser. 24, 197-200). A rapidly growing area that uses nucleic acid hybridisation for direct detection of nucleic acid sequences is that of DNA microarrays, (2002, Nature Genetics, 32, [Supplement]; 2004, Cope, L. M., et al., Bioinformatics, 20, 323-331; 2004, Kendall, S. L., et al., Trends in Microbiology, 12, 537-544). In this process, individual nucleic acid species, that may range from short oligonucleotides, (typically 25-mers in the Affymetrix system), to longer oligonucleotides, (typically 60-mers in the Applied Biosystems and Agilent platforms), to even longer sequences such as cDNA clones, are fixed to a solid support in a grid pattern or photolithographically synthesized on a solid support. A tagged or labelled nucleic acid population is then hybridised with the array and the level of hybridisation to each spot in the array quantified. Most commonly, radioactively- or fluorescently-labelled nucleic acids (eg cRNAs or cDNAs) are used for hybridisation, though other detection systems can be employed, such as chemiluminescence.
Currently, there is much interest in harnessing molecular methods for the diagnosis of infectious disease, since such newer methods hold the promise of sensitive and specific detection of pathogenic organisms. In this context, the present invention deals with human papilloma virus (HPV), whose DNA genome exists at the populational level as a variable gene pool with individual HPV types differing both at the nucleotide sequence level as well as in the sizes of their genomes. Detecting and accurately identifying different HPV types in various clinical samples via molecular tests is hampered by the limitations of the various molecular tests. In addition, a large number of ‘genotypes’, ‘variants’, ‘subtypes’ and ‘types’ exist within the umbrella grouping that defines HPV. For example, there are now over 100 recognized types of HPV some of which are strongly correlated with human disease. The so called high- and medium-risk types are implicated in the progression to cancer and their detection via the most accurately available molecular methods is an urgent clinical priority.
The major problem is that detection of HPV alone is not necessarily a very good indicator of progression to cancer. Although Cervical Intraepithelial Neoplasia (CIN) can progress to an invasive form, many lesions either regress or persist but without progressing to carcinoma. Seventy percent of women will clear an HPV infection within two years (1998, Journal of Pediatrics, 132, 277-284; Moscicki, A. B., et al.). However, the finding of CIN and its progression is so variable that even untreated, it may return to normalcy or lead to a full blown carcinoma. Approximately one third to one half of CINI and CINII cases spontaneously regress (1990, Australian and New Zealand Journal of Obstetrics and Gynaecology, 30, 1-23., Channen, W et al.), The time taken to progress from CINI to CINII is of the order of a decade, but in some females can be two decades or more. (2000, Cancer Research, 60, 6027-6032., Ylitalo, E et al.).
Viral infection of a cell, tissue or organ can cause the genome methylation signature of these entities to be altered, as in the case of Epstein-Barr virus and it's association with gastric carcinoma (2002, American J. Pathology, 160, 787-794, Kang, G. H., et al). Since methylation or demethylation is a stable change inherited over many cell divisions, and in some cases, generations of organisms, an alteration of the usually stable methylome can be predictive of the pre-cancerous or cancerous state.
It has been challenging to implement reliable and robust DNA-based detection systems that recognise all the different HPV types in a single assay, since not only are there cross hybridization problems between different HPV genomic types, but the exact classification of what constitutes an HPV type is dependent upon genomic sequence similarities which have significant bioinformatic limitations. Thus, while new HPV types have been defined as ones where there is less than 90% sequence similarity with previous HPV types, finer taxonomic subdivisions are more problematic to deal with. Thus, a new HPV ‘subtype’ is defined when the DNA sequence similarity is in the 90-98% range relative to previous subtypes. A new ‘variant’ is defined when the sequence similarity is between 98-100% of previous variants (1993, Van Rast, M. A., et al., Papillomavirus Rep, 4, 61-65; 1998, Southern, S. A. and Herrington, C. S. Sex. Transm. Inf. 74, 101-109). This spectrum can broaden further to the point where variation could be measured based on comparing single genomes from single isolated viral particles. In such a case, a ‘genotype’ would be any fully sequenced HPV genome that minimally differs by one base from any other fully sequenced HPV genome. This includes all cases where a single base at a defined position can exist in one of four states, G, A, T or C, as well as cases where the base at that given position has been altered by deletion, addition, amplification or transposition to another site.
For the above reasons, all the bioinformatic comparisons used in the present patent specification application are made relative to the HPV16 genome (using positions 1 to 7904 of HPV16 as the standard comparator), and using prior art BLAST methodologies, (1996, Morgenstern, B., et al., Proc. Natl. Acad. Sci. USA. 93, 12098-12103). The standard HPV ‘type’ utilized herein for reference purposes is HPV16 of the Papillomaviridae, a papillomavirus of 7904 base pairs (National Center for Biotechnology Information, NCBI locus NC—001526; version NC—001526.1; GI:9627100; references, Medline, 91162763 and 85246220; PubMed 1848319 and 2990099).
The difficulties faced by existing HPV detection systems in the context of disease risk assessment are largely threefold. First limitations of the technology systems themselves. Secondly, limitations of the pathological interpretations of diseased cell populations. Thirdly, limitations at the clinical level of assessing disease progression in different human populations that are subject to differences in genetic background as well as contributing cofactors.
HPV of certain types are implicated in cancers of the cervix and contribute to a more poorly defined fraction of cancers of the vagina, vulvae, penis and anus. The ring of tissue that is the cervical transformation zone is an area of high susceptibility to HPV carcinogenicity, and assessment of its state from complete cellular normalcy to invasive carcinoma has been routinely evaluated using visual or microscopic criteria via histological, cytological and molecular biological methodologies. The early detection of virally-induced abnormalities at both the viral level and that of the compromised human cell, would be of enormous clinical relevance if it could help in determining where along a molecular trajectory, from normal to abnormal tissue, a population of cells has reached. However, despite the use of the Pap smear for half a century, a solid early risk assessment between abnormal cervical cytological diagnoses and normalcy is currently still problematical. Major problems revolve around the elusive criteria on which to define ‘precancer’, such as the various grades of Cervical Intraepithelial Neoplasia, (CIN1, CIN2 and CIN3) and hence on the clinical decisions that relate to treatment options. Precancer definitions are considered by some clinicians to be a pseudo-precise way in which to avoid using CIN2, CIN3 and carcinoma in situ. There is great heterogeneity in microscopic diagnoses and even in the clinical meaning of CIN2, (2003, Schiffman, M., J. Nat. Cancer Instit. Monog. 31, 14-19). Some CIN2 lesions have a bad microscopic appearance but will nevertheless be overcome by the immune system and disappear, whereas other lesions will progress to invasive carcinoma. Thus CIN2 is considered by some as a buffer zone of equivocal diagnosis although the boundary conditions of such a zone remain controversial. Some clinicians consider it to be poor practice to combine CIN2 and CIN3, whereas others will treat all lesions of CIN2 or worse. Finally, the literature indicates that between a third and two thirds of CIN3 assigned women will develop invasive carcinoma, but even this occurs in an unpredictable time-dependent fashion, (2003, Schiffman, M., J. Nat. Cancer. Instit. Monog. 31, 14-19; 1978, Kinlen, L. J., et al., Lancet 2, 463-465; 1956, Peterson, O. Am. J. Obstet. Gynec. 72, 1063-1071).
The central problem still confronting physicians today is that defining low grade cytological abnormalities such as atypical squamous cells of undetermined significance, (ASCUS), or squamous intraepithelial lesions (SILs) is difficult. ‘In fact, ASCUS is not a proper diagnosis but rather is a “wastebasket” category of poorly understood changes’, (1996, Lorincz, A. T., 1996, J. Obstet. Gyncol. Res. 22, 629-636). The whole spectrum of precancerous lesions is difficult to interpret owing to cofactor effects from oral contraceptive use, smoking, pathogens other than HPV such as Chlamydia trachomatis and Herpes Simplex Virus type 2, antioxidant nutrients and cervical inflammation, all of which are claimed to modulate the risk of progression from high grade squamous intraepithelial lesions (HSILs) to cervical cancer (2003, Castellsague, X. J. Nat. Cancer Inst. Monog. 31, 20-28). The introduction of the Bethesda system of classification and its revision in 2001 has done little to reduce the confusion among clinicians, since it was initially found unhelpful to include koilocytotic atypia with CIN1 into the newer category of low-grade squamous intraepithelial lesions, (LSILs). The result of the introduction of the Bethesda system was that many clinicians would not carry out colposcopy on koilocytotic atypia, but felt compelled do so on patients with CIN1′, (1995, Hatch, K. D., Am. J. Obstet. Gyn. 172, 1150-1157). It was clear that although colposcopic expertise required many years of training, subjective cytological criteria still lead to inconsistencies and non-reproducibilities, (1994, Sherman, M. E., Am. J. Clin. Pathology, 102, 182-187; 1988, Giles, J. A., Br. Med. J., 296, 1099-1102).
The continuing diagnostic hurdle is that vague diagnoses such as ‘atypia’ can account for 20% or more of diagnoses in some settings, (1993, Schiffman, M. Contemporary OB/GYN, 27-40). This is illustrated by a test designed specifically to evaluate the level of independent diagnostic agreement of pathologists on smears that were ‘atypical’. It was found that exact agreement between five professional pathologists on an identical set of samples occurred in only 29% of cases, (1994, Sherman, M. E., et al., Am. J. Clin. Pathology, 102, 182-187). The net result is that cervical cytology continues to have high false negative rates (termed low sensitivity) and high false positive rates, (termed low specificity). The cytological interpretations of various pathologists yield a false negative rate of up to 20% or so and a false positive rate of up to 15% (1993, Koss, L. G., Cancer, 71, 1406-1412). False positive results lead to unnecessary colposcopic examinations, biopsies and treatments, all of which add to the health care cost burden. False negative results lead to potential malpractice law suits with their associated costs. It was into this arena that molecular diagnoses of early stages of cervical abnormalities using tests for HPV offer a less subjective test than cytological ones.
Genomic indicators of a lack of well being in an organism are intimately tied to changes in genomic methylation status at a number of levels. Dietary supplementation can have unintended and deleterious consequences on methylation and metabolic well being, (Waterland, R. A., 2003, Molecular and Cellular Biology, 23, 5293-5300) and aberrant methylation of certain genomic promoter regions, such as that of the reelin locus, are implicated in various psychiatric conditions such as schizophrenia (2002, Chen, Y., et al., Nucleic Acids Research, 30, 2930-2939; Miklos, G. L. G., and Maleszka, R., 2004, Nature Biotechnology, 22, 615-621). The single largest area of methylation investigation is in cancer research, where both hypermethylation and hypomethylation of genomic regions, is extensively documented (French, S. W., et al., 2002, Clinical Immunology, 103, 217-230; Frigola, J., et al., 2005, Human Molecular Genetics, 14, 319-326; Belinsky S. A., et al., Nature Reviews Cancer, 4, 1-11). Some of these studies aim at uncovering prognostic biomarkers (Baker, M., 2005, Nature Biotechnology, 23, 297-304) for indications of cancer, but the biomarker field is riddled with inconsistent results. In addition, rarely are such genomic studies interfaced with other sources of perturbations to cells and tissues that arise from infections with microorganisms and viruses. In addition, cancer genomes generally contain massive genomic upheavals, such as chromosomal aneuploidy, segmental aneuploidy, deletions, amplifications, inversions, translocations and multiple mutations (Duesberg, P., 2004, Cell Cycle, 3, 823-828; Miklos, G. L. G., 2005, Nature Biotechnology, 23, 535-537) and the importance of these for early detection, in the context of methylation changes, has not yet been very deeply explored, (Vogelstein, B., et al., 2004, Nature Medicine, 10, 789-799; Lucito, R., at al., 2003, Genome Research, 13, 2291-2305).
Given all the problems and shortcomings outlined above, there is still controversy as regards the clinical impact of DNA methodologies particularly in screening for pre-neoplastic lesions. Sensitive early molecular prognostic indicators of cellular abnormalities would be extremely valuable. The present inventors have developed new methods, kits and integrated bioinformatic platforms for detecting viruses and genomic targets for use in determining the health state of an individual.